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The Babesia divergens/B. capreoli group includes parasites with confirmed or possible zoonotic potential to cause human babesiosis. Currently, diagnostic antigen of the group has not been established. In this study, we investigated the ortholog of Bd37, a glycosylphosphatidylinositol (GPI)-anchored major merozoite surface protein of B. divergens sensu stricto, in the Asia lineage of the group. From two genomic isolates from sporozoite/sporoblast stages, three Bd37 gene variants, namely Bd37 JP-A, JP-B, and JP-C, were isolated with 62.3-64.1% amino acid sequence identity. Discriminative blood direct PCR revealed that Bd37 JP-A was encoded in all parasites infecting wild sika deer examined (n=22). While Bd37 JP-B and JP-C genes were randomly detected in 12 and 11 specimens, respectively. Sequencing of all JP-A variants revealed that the gene was polymorphic, with a low ratio of non-synonymous to synonymous substitutions (dN/dS) and that a highly polymorphic region was not related to predicted B-cell epitopes. A recombinant JP-A-based ELISA showed an overall positive rate of 13.9% in sika deer in Japan from north (Hokkaido) to south (Kyushu island) across 24 prefectures (n=360). This positive rate was twice as high as that examined by 18S rRNA-based PCR (6.6%). The geographical trends in infection rates were consistent. This study demonstrated that direct examination was informative for revealing genetic background and selecting antigen candidates. Bd37 orthologs may serve diagnostic purposes in combination with indirect fluorescence assay, which requires biological isolates.

The fireworms Odontosyllis spp. are globally distributed and well-known for their characteristic and fascinating mating behavior, with secreted mucus emitting bluish-green light. However, knowledge about the molecules involved in the light emission are still scarce. The fireworms are believed to emit light with a luciferin-luciferase reaction, but biochemical evidence of the luciferase is established for only one species living in Japan and no information is available for its luciferin structure. In this study, we identified a luciferase gene from a related Puerto Rican fireworm. We identified eight luciferase-like genes in this Puerto Rican fireworm, finding amino acid identities between Japanese and Puerto Rican luciferase-like genes to be less than 60%. We confirmed cross reactivity of extracts of the Japanese fireworm luciferin with a recombinant Puerto Rican luciferase (PR1). The emission spectrum of recombinant PR1 was similar to the crude extract of the native luciferase, suggesting that PR1 is a functional luciferase of this Puerto Rican fireworm. Our results indicate that the molecular mechanism of luminescence is widely conserved among fireworms.

Background: Conditioning chemotherapies for hematopoietic stem cell transplantation (HSCT), especially those that include total body irradiation, can result in serious complications such as graft-versus-host disease (GVHD). Human leukocyte antigen G (HLA-G) is a non-classical class I molecule with multiple immunoregulatory functions., Methods: We measured interleukin (IL)-10, transforming growth factor (TGF)β 1 , and soluble HLA-G (sHLA-G) in HSCT patients and examined the relationship between sHLA-G levels and acute GVHD (aGVHD). Additionally, we investigated the effect of recombinant soluble thrombomodulin (rTM) therapy on sHLA-G levels. Our study cohort included 135 patients who underwent allogeneic HSCT at several institutions in Japan., Results: Serum levels of IL-10 and TGFβ 1 exhibited no significant changes following HSCT. In contrast, levels of sHLA-G were significantly increased at days 21 and 28 post-HSCT. For patients with confirmed complications, the frequency of aGVHD was significantly lower in those with a > 2.8-fold increase in sHLA-G levels at day 28 relative to day 7 post-HSCT. sHLA-G levels in patients who received rTM therapy were significantly higher at days 21 and 28 post-HSCT compared with those in patients who did not receive rTM therapy., Conclusion: These data suggest that HLA-G/sHLA-G participate in prevention of GVHD, and that rTM may prevent aGVHD following HSCT by promoting elevation of sHLA-G., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Recombinant human soluble thrombomodulin (rTM) is a newly developed anti-coagulant approved for treatment of disseminated intravascular coagulation (DIC) in Japan. rTM exerts anti-inflammatory and cytoprotective functions via its lectin-like and epidermal growth factor-like domains, respectively. In this study, we retrospectively reviewed the treatment of 21 consecutive patients with coagulopathy, complicated by acute promyelocytic leukemia (APL), with all-trans retinoic acid (ATRA) with or without combination with rTM. Surprisingly, none of the 14 rTM-treated patients developed retinoic acid (RA)-related differentiation syndrome (DS). The co-culture of vascular endothelial cell-derived EA.hy926 and APL-derived NB4 cells in the presence of RA increased production of tumor necrosis factor alpha (TNF-α) in culture media, in parallel with activation of p38 mitogen-activated protein kinase and increased levels of intracellular adhesion molecule 1 (ICAM1) in EA.hy926 cells. This was also associated with increased levels of the phosphorylated forms of VE-cadherin and enhanced vascular permeability of EA.hy926 monolayers. Importantly, addition of rTM to this co-culture system inhibited the RA-induced phosphorylation of p38 and VE-cadherin and decreased ICAM1 and vascular permeability in EA.hy926 cells, without a decrease inthe levels of TNF-α. Taken together, use of rTM may be a promising treatment strategy to prevent DS in APL patients who receive ATRA., (Copyright © 2017. Published by Elsevier Ltd.)

Japan has only three registered baculovirus biopesticides despite its long history of studies on insect viruses. High production cost is one of the main hindrances for practical use of baculoviruses. Enhancement of insecticidal effect is one possible way to overcome this problem, so there have been many attempts to develop additives for baculoviruses. We found that alkaline soluble proteins of capsules (GVPs) of Xestia c-nigrum granulovirus can increase infectivity of some viruses including Mamestra brassicae nucleopolyhedrovirus (MabrNPV), and previously reported that MabrNPV mixed with GVPs was highly infectious to three important noctuid pests of vegetables in the following order, Helicoverpa armigera, M. brassicae, and Autographa nigrisigna. In this study, small-plot experiments were performed to assess concentrations of MabrNPV and GVPs at three cabbage fields and a broccoli field for the control of M. brassicae. In the first experiment, addition of GVPs (10 µg/mL) to MabrNPV at 106 OBs/mL resulted in a significant increase in NPV infection (from 53% to 66%). In the second experiment, the enhancing effect of GVP on NPV infection was confirmed at 10-times lower concentrations of MabrNPV. In the third and fourth experiments, a 50% reduction in GVPs (from 10 µg/mL to 5 µg/mL) did not result in a lowering of infectivity of the formulations containing MabrNPV at 105 OBs/mL. These results indicate that GVPs are promising additives for virus insecticides.

A hyperthermophilic archaeon was isolated from a terrestrial hot spring on Kodakara Island, Japan and designated as Thermoproteus sp. glucose dehydrogenase (GDH-1). Cell extracts from cells grown in medium supplemented with glucose exhibited NAD(P)-dependent glucose dehydrogenase activity. The enzyme (TgGDH) was purified and found to display a strict preference for D-glucose. The gene was cloned and expressed in Escherichia coli, resulting in the production of a soluble and active protein. Recombinant TgGDH displayed extremely high thermostability and an optimal temperature higher than 85 °C, in addition to its strict specificity for D-glucose. Despite its thermophilic nature, TgGDH still exhibited activity at 25 °C. We confirmed that the enzyme could be applied for glucose measurements at ambient temperatures, suggesting a potential of the enzyme for use in measurements in blood samples.

Pleurocybella porrigens is a mushroom-forming fungus, which had been consumed as a traditional food in Japan. However, in 2004, 55 people got poisoned by eating the mushroom and 17 people among them died of acute encephalopathy. We have already reported the purification, characterization, and cDNA cloning of a lectin from the mushroom (PPL) which might have caused the poisoning. Here, we report the heterologous expression of recombinant PPL by basidiomycete Phanerochaete sordida YK-624. The glyceraldehyde 3-phosphate dehydrogenase gene promoter was used to drive the expression of the PPL gene (ppl) in P. sordida YK-624. Furthermore, the signal peptide of lignin peroxidase which is an extracellular protein was used to secrete rPPL into extracellular region. Fifteen regenerated clones were cultured on Kirk HNHC broth, and the presence of lectin activity in the culture broth was checked by agglutination assays. The results indicated that the culture broth of rPPL-6 clone showed the strongest hemagglutination activity, and it was therefore used for subsequent analysis. The heterologous expression of rPPL by P. sordida YK-624 was confirmed by SDS-PAGE, lectin activity by the hemagglutination assay, and mass of rPPL by MALDI-TOF respectively, indicating that the extracellular secretion of rPPL as active form was successful., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We describe here isolation of genetically atypical serotype 6 Actinobacillus pleuropneumoniae in Japan indistinguishable by the multiplex PCR that can discriminate between immunologically cross-reactive serotypes 3, 6 and 8. Nucleotide sequence analysis of capsular export and biosynthesis genes revealed that the atypical isolates have capsular polysaccharide export and synthesis gene sequences that are distinct from those of the serotype 6 reference strain. The atypical strains contain a sequence that is identical with both serotype 3- and 6-specific primers, which causes cross-reactions in multiplex PCR.

Salmon roe has a high allergic potency and often causes anaphylaxis in Japan. The major allergic protein of salmon roe is β'-component, which is a 35kDa vitellogenin fragment consisting of two subunits. To elucidate structural information and immunological characteristics, β'-component and the subunit components were purified from chum salmon (Onchorhincus keta) roe and vitellogenin-encoding mRNA was used to prepare β'-component subunit-encoding cDNA. This was PCR-amplified, cloned and sequenced and the deduced amino acid sequence compared with partial sequences of β'-component obtained by peptide mapping. The recombinant β'-component subunit was produced by bacterial expression in Escherichia coli and its IgE-binding ability was measured by ELISA using the sera of a patient allergic to salmon roe. This was then compared with that of the native β'-component with and without carboxymethylation. Following successful cloning of the cDNA encoding the β'-component subunit, 170 amino acid residues were deduced and matched with the amino acid sequences of 121 and 88 residues in the 16kDa and 18kDa subunits, respectively. The sequences of both β'-component subunits were almost identical, and the predicted secondary structure of the β'-component showed a high content of β-pleated sheets and no α-helices. There was no difference in IgE-binding ability between the native and recombinant β'-component subunits at the same protein concentration, regardless of carboxymethylation. In conclusion, β'-component is a homodimer protein composed of two isoform subunits having the same level of IgE-binding ability and, therefore, allergenic identity.

The levels of diversity and extent of linkage disequilibrium in cultivated species are largely determined by diversity in their wild progenitors. We report a comparison of nucleotide sequence diversity in wild and cultivated barley (Hordeum vulgare ssp. spontaneum and ssp. vulgare) at 7 nuclear loci totaling 9296bp, using sequence from Hordeum bulbosum to infer the ancestral state of mutations. The sample includes 36 accessions of cultivated barley, including 23 landraces (cultivated forms not subject to modern breeding) and 13 cultivated lines and genetic stocks compared to either 25 or 45 accessions of wild barley for the same loci. Estimates of nucleotide sequence diversity indicate that landraces retain >80% of the diversity in wild barley. The primary population structure in wild barley, which divides the species into eastern and western populations, is reflected in significant differentiation at all loci in wild accessions and at 3 of 7 loci in landraces. "Oriental" landraces have slightly higher diversity than "Occidental" landraces. Genetic assignment suggests more admixture from Occidental landraces into Oriental landraces than the converse, which may explain this difference. Based on θπ for silent sites, modern western cultivars have ~73% of the diversity found in landraces and ~71% of the diversity in wild barley.

A member of the NF-κB signaling pathway, PoAkirin1, was cloned from a full-length cDNA library of Japanese flounder (Paralichthys olivaceus). The full-length cDNA comprises a 5'UTR of 202 bp, an open reading frame of 564 bp encoding a 187-amino-acid polypeptide and a 521-bp 3'UTR with a poly (A) tail. The putative protein has a predicted molecular mass of 21 kDa and an isoelectric point (pI) of 9.22. Amino acid sequence alignments showed that PoAkirin1 was 99% identical to the Scophthalmus maximus Akirin protein (ADK27484). Yeast two-hybrid assays identified two proteins that interact with PoAkirin1: PoHEPN and PoC1q. The cDNA sequences of PoHEPN and PoC1q are 672 bp and 528 bp, respectively. Real-time quantitative reverse-transcriptase polymerase chain reaction analysis showed that bacteria could induce the expressions of PoAkirin1, PoHEPN and PoC1q. However, the responses of PoHEPN and PoC1q to the bacterial challenge were slower than that of PoAkirin1. To further study the function of PoAkirin1, recombinant PoAkirin1 and PoHEPN were expressed in Escherichia coli and would be used to verify the PoAkirin1-PoHEPN binding activity. These results identified two proteins that potentially interact with PoAkirin1 and that bacteria could induce their expression.

We isolated cDNA for a luciferase isotype, LlLuc2, from the ovary of the Japanese firefly, Luciola lateralis. The gene product LlLuc2 showed 59% amino acid identity with LlLuc1, which had been isolated from the adult L. lateralis lantern. Molecular phylogenetic analysis indicated that LlLuc2 is an orthologue of LcLuc2 from Luciola cruciata. The spectral maxima of the luminescence by recombinant LlLuc1 and LlLuc2 were 550 and 539 nm, respectively. Quantitative PCR analysis revealed that LlLuc1 was expressed predominantly in larvae and adults, and LlLuc2 was expressed in eggs and pupae, which glow dimly, and we found that the in vivo luminescence spectra of the egg and pupa in L. lateralis were in good agreement with the in vitro luminescence spectrum by LlLuc2. These results suggest that, in L. lateralis, LlLuc1 is responsible for the yellowish luminescence of larval and adult lanterns, and LlLuc2 is responsible for the dim, greenish glow of eggs and whole pupae. Similar results were obtained in L. cruciata.

In the present study, we investigated the possible tick vectors that can transmit Theileria orientalis in eastern Hokkaido, Japan. Questing ticks collected from three different districts, Taiki, Otofuke, and Shin-Hidaka, of Hokkaido included Ixodes persulcatus, Haemaphysalis megaspinosa, Haemaphysalis douglasi, and Ixodes ovatus, while all the ticks collected from Yonaguni island of Okinawa were identified as Haemaphysalis longicornis. When the ticks were screened by polymerase chain reaction (PCR) for T. orientalis, the parasite was commonly detected among all tick species. Genotype-specific PCR assays revealed that all tick species in Hokkaido were predominantly detected with type 2, while ticks collected from Okinawa (H. longicornis) were predominantly detected with type 1. Consistent with the genetic diversity of T. orientalis in ticks, genotyping PCR assays from cattle grazed in the same Hokkaido sampling locations identified type 2 as the most prevalent genotype. This study provides the first identification of I. persulcatus, H. megaspinosa, H. douglasi, and I. ovatus as possible tick vectors of T. orientalis, and finds that the variety of vectors apparently capable of transmitting T. orientalis is wider in Japan than expected. The authors suggest that tick control strategies should be modified in Hokkaido based on the seasonal activities of ticks identified in the present study., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Loss-of-function mutations of flavin-containing monooxygenase 3 (FMO3), the enzyme responsible for trimethylamine N-oxygenation, cause the inherited disorder trimethylaminuria, or fish odor syndrome. The aim of this study was to further investigate the inter-individual variations of FMO3 activity in a Japanese cohort that we had studied previously. The subjects were 640 Japanese volunteers with self-reported trimethylaminuria; genomic DNA was sequenced in those that had 10-70% FMO3 metabolic capacity in urine tests. A heterozygote for the novel single nucleotide substitution p.Ile441Thr (proband 1) and a heterozygote for the novel single nucleotide substitution p.Ser195Leu (proband 2) were identified. The biological parents of probands 1 and 2 were heterozygous and had >90% trimethylamine N-oxygenation metabolic capacity. In addition, single nucleotide substitutions p.Val58Ile, p.Pro70Leu, and p.Gly421Val in FMO3 were found in probands 3-7. In the course of DNA sequencing, another FMO3 variant, p.Thr488Ala, was found in two unrelated heterozygous subjects. Variant FMO3 proteins recombinantly expressed in Escherichia coli membranes exhibited decreased activity toward typical FMO3 substrates. Although the allele frequencies of these six novel variants were low (<1%), the present results suggest that individuals homozygous or heterozygous for any of the six novel missense FMO3 variants or known nonsense mutations such as p.Cys197stop or p.Arg500stop may possess abnormal trimethylamine N-oxygenation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Chitiniphilus shinanonensis type strain SAY3(T) is a strongly chitinolytic bacterium, originally isolated from the moat water in Ueda, Japan. To elucidate the chitinolytic activity of this strain, 15 genes (chiA-chiO) coding for putative chitin-degrading enzymes were isolated from a genomic library. Sequence analysis revealed the genes comprised 12 family 18 chitinases, a family 19 chitinase, a family 20 β-N-acetylglucosaminidase, and a polypeptide with a chitin-binding domain but devoid of a catalytic domain. Two operons were detected among the sequences: chiCDEFG and chiLM. The gene coding for the polypeptide (chiN) showed sequence similarity to family 19 chitinases and was successfully expressed in Escherichia coli. ChiN demonstrated a multi-domain structure, composed of the N-terminal, two chitin-binding domains connected by a Pro- and Thr-rich linker, and a family 19 catalytic domain located at the C-terminus. The recombinant protein rChiN catalyzed an endo-type cleavage of N-acetyl-d-glucosamine oligomers, and also degraded insoluble chitin and soluble chitosan (degree of deacetylation of 80%). rChiN exhibited an inhibitory effect on hyphal growth of the fungus Trichoderma reesei. The chitin-binding domains of ChiN likely play an important role in the degradation of insoluble chitin, and are responsible for a growth inhibitory effect on fungi., (Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

We performed gene-based comparative FISH mapping between a wild silkmoth, Samia cynthia ssp. with a low number of chromosomes (2n=25-28) and the model species, Bombyx mori (2n=56), in order to identify the genomic components that make up the chromosomes in a low-number karyotype. Mapping of 64 fosmid probes containing orthologs of B. mori genes revealed that the homologues of either two or four B. mori chromosomes constitute the S. c. ricini (Vietnam population, 2n=27♀/28♂, Z0/ZZ) autosomes. Where tested, even the gene order was conserved between S. c. ricini and B. mori. This was also true for the originally autosomal parts of the neo-sex chromosomes in S. c. walkeri (Sapporo population, 2n=26♀/26♂, neo-Wneo-Z/neo-Zneo-Z) and S. cynthia subsp. indet. (Nagano population, 2n=25♀/26♂, neo-WZ₁Z₂/Z₁Z₁Z₂Z₂). The results are evidence for an internal stability of lepidopteran chromosomes even when all autosomes had undergone fusion processes to form a low-number karyotype., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: Xanthine oxidase (XO) catalyzes the oxidation of endogenous and exogenous purines and pyrimidines. In this study, we speculated that individual variations in XO activity are caused by genetic variations in the XO gene., Methods: To investigate the genetic variations in XO in 96 Japanese participants, denaturing high-performance liquid chromatography was used. To assess the effects of these variations on enzymatic activity, wild-type XO and 21 types of variant XO--including those in the database and those just discovered--were transiently expressed in COS-7 cells., Results: Three nonsynonymous single nucleotide polymorphisms, including 514G>A (Gly172Arg), 3326A>C (Asp1109Thr), and 3662A>G (His1221Arg) were identified in Japanese participants. Functional characterization of 21 XO variants showed a deficiency in enzyme activity in two variants (Arg149Cys and Thr910Lys); low activity (intrinsic clearance, CLint: 22-69% compared with the wild-type) in six variants (Pro555Ser, Arg607Gln, Thr623Ile, Asn909Lys, Pro1150Arg, and Cys1318Tyr); and high activity (CLint: approximately two-fold higher than that in the wild-type) in two variants (Ile703Val and His1221Arg)., Conclusion: These results suggest that several single nucleotide polymorphisms in the XO gene are involved in individual variations in XO activity. In addition, such findings will be useful to identify xanthinuria patients.

Background: Uteroglobin-related protein 1 (UGRP1) is a secretory protein expressed in the airways and is speculated to have anti-inflammatory activity. In the mouse, its gene expression is down-regulated by interleukin (IL)-5 and -9, and up-regulated by IL-10. However, the precise role of UGRP1 in human inflammatory airway diseases such as asthma has not been clarified. The objectives of this study were to establish an ELISA system to quantify UGRP1 protein, and to examine whether plasma UGRP1 levels are associated with the G-112A polymorphism, asthma susceptibility, and its severity., Methods: 152 asthma patients and 103 normal controls were involved in this study. Mice were immunized with recombinant UGRP1 and hybridoma cell lines were established. A sandwich ELISA system was established by using two monoclonal antibodies recognizing different epitopes. Plasma UGRP1 levels were measured with the ELISA system and the G-112A allele was detected by using real-time PCR., Results: An ELISA system was established that allowed determination of UGRP1 levels within the range of 9.6-1250 pg/ml. The mean plasma UGRP1 levels for subjects with -112A allele were significantly lower than those without it (p = 0.025). Although there was no significant difference in the plasma UGRP1 levels between asthma patients and controls (p = 0.13), severe asthma patients without oral corticosteroid had significantly lower plasma UGRP1 levels compared to mild- or moderate- asthma patients and controls (p = 0.004, 0.03 and 0.003, respectively)., Conclusions: The ELISA system for quantifying UGRP1 protein was established, and plasma UGRP1 levels were associated with the G-112A UGRP1 gene promoter polymorphism and the severity of asthma.

Background: Birch pollen is the major allergen in pollinosis in northern Japan. IgE reactivity to individual birch pollen allergens has been shown to differ between populations of birch pollen-allergic patients living in different countries. In this study, we examined the IgE profiles to recombinant birch pollen allergens in birch-sensitive patients living in Sapporo., Methods: This study used the sera of 40 patients with specific IgE toward birch pollen extract. Their sera were analyzed for specific IgE reactivity to individual birch pollen allergens (recombinant Bet v 1, Bet v 2 and Bet v 4) and natural birch pollen extract using Pharmacia CAP SystemTM., Results: Of 40 sera with positive CAP results for natural birch pollen extract, 39 (97.5%) had specific IgE towards Bet v 1; 6 (15%) contained specific IgE against Bet v 2. Bet v 4 reactivity was documented in only one subject (2.5%)., Conclusions: The present data suggest that the specific IgE reactivity profiles to birch pollen allergen in birch-sensitive patients in Sapporo correspond to those in Scandinavia, possibly due to the heavy birch pollen exposure in this area. This observation provides useful information for future birch allergen-specific immunotherapy in Japan.

The NADase (Nga) of group A streptococci (GAS) has been implicated in the pathogenesis of diseases such as streptococcal toxic shock-like syndrome (STSS) and necrotizing fasciitis. In this study we found that the proportion of NADase-positive strains among clinical isolates in Japan has increased over time. The GAS strains studied could be divided into three groups: strains lacking NADase activity, strains with low NADase activity, and strains with high NADase activity. The older strains, isolated before 1989, belonged to the 'no activity' group. Analysis using GST-Nga recombinants revealed that nga alleles of representative older strains encode inactive Nga. Mutational analysis of the GST-Nga recombinants suggested that residue 330 could be associated with reduced activity, based upon deduced amino acid sequences. We also investigated NADase activity of streptococcal strains other than GAS. All group G streptococcal isolates from STSS patients possessed nga genes encoding active enzymes.

Background: We are developing an immunotherapeutic peptide, Cry-consensus peptide, for Japanese cedar pollinosis. Cry-consensus peptide is a recombinant polypeptide containing six major human T-cell epitopes derived from both Cry j 1 and Cry j 2, two major allergens of Japanese cedar pollen. We examined the effect of Cry-consensus peptide on an allergic rhinitis model in B10.S mice, which have one common T-cell epitope in the Cry-consensus peptide., Methods: B10.S mice were sensitized with Cry j 1/alum, then the Cry-consensus peptide was administered subcutaneously once a week for 5 weeks from the last sensitization. Histamine was dropped in both nostrils (10 microL per nostril) of each mouse on the day before continuous intranasal instillation of Cry j 1. Soon after the final challenge with Cry j 1, the mice were observed for 5 minutes for the resulting number of sneezes. In addition, serum levels of Cry j 1-specific IgE and IgG2a antibody, eosinophil infiltration in nasal tissue, and Cry j 1-specific cytokine production from splenocytes were evaluated., Results: Cry-consensus peptide markedly inhibited Cry j 1-induced sneezes, eosinophil infiltration, and eosinophil peroxidase (EPO) activity in nasal tissue. Cry-consensus peptide inhibited the production of anti-Cry j 1 IgE (Th2-mediated) and significantly enhanced anti-Cry j 1 IgG2a (Th1-mediated). In cytokine production from splenocytes, Cry-consensus peptide significantly decreased in IL-4/IFN-gamma and IL-5/IFN-gamma ratios., Conclusions: It was concluded that Cry-consensus peptide effectively controlled allergic responses, which results from shifting from a Th2-dominated to a Th1-dominated immune response.

Cytochrome P450 2C19 (CYP2C19) plays an important role in the metabolism of a wide range of therapeutic drugs and exhibits genetic polymorphism with interindividual differences in metabolic activity. We have previously described two CYP2C19 allelic variants, namely CYP2C19*18 and CYP2C19*19 with Arg329His/Ile331Val and Ser51Gly/Ile331Val substitutions, respectively. In order to investigate precisely the effect of amino acid substitutions on CYP2C19 function, CYP2C19 proteins of the wild-type (CYP2C19.1B having Ile331Val) and variants (CYP2C19.18 and CYP2C19.19) were heterologously expressed in yeast cells, and their S-mephenytoin 4'-hydroxylation activities were determined. The K(m) value of CYP2C19.19 for S-mephenytoin 4'-hydroxylation was significantly higher (3.0-fold) than that of CYP2C19.1B. Although no significant differences in V(max) values on the basis of microsomal and functional CYP protein levels were observed between CYP2C19.1B and CYP2C19.19, the V(max)/K(m) values of CYP2C19.19 were significantly reduced to 29-47% of CYP2C19.1B. By contrast, the K(m), V(max) or V(max)/K(m) values of CYP2C19.18 were similar to those of CYP2C19.1B. These results suggest that Ser51Gly substitution in CYP2C19.19 decreases the affinity toward S-mephenytoin of CYP2C19 enzyme, and imply that the genetic polymorphism of CYP2C19*19 also causes variations in the clinical response to drugs metabolized by CYP2C19.

Background: IL-17F is a recently discovered cytokine that plays a role in tissue inflammation by inducing release of proinflammatory and neutrophil-mobilizing cytokines. Upregulated IL17F gene expression has been observed at sites of allergen challenge in the airways of patients with asthma, suggesting that IL-17F is involved in the pathophysiology of asthma., Objective: To investigate the role of IL-17F in asthma pathogenesis, we conducted genetic analyses of association of asthma with the common variants of IL17F, using 867 unrelated Japanese subjects., Methods: Five polymorphisms were studied, including the coding-region sequence variant single nucleotide polymorphism rs763780 (7488T/C), which causes a His-to-Arg substitution at amino acid 161 (H161R). Functional consequences of the H161R substitution were examined by using recombinant wild-type and mutant IL-17F proteins., Results: Homozygosity of the H161R variant was inversely associated with asthma; the odds ratio (95% CI) for asthma was 0.06 (0.01-0.43) for the H161R homozygote compared with the wild-type homozygote (P = .0039). This result remains significant (P = .0079) after adjustment for the presence of atopy using the Mantel-Haenszel chi2 test. In addition, in vitro functional experiments demonstrated that the H161R variant of IL-17F lacks the ability to activate the mitogen-activated protein kinase pathway, cytokine production, and chemokine production in bronchial epithelial cells, unlike wild-type IL-17F. Furthermore, the H161R variant blocked induction of IL-8 expression by wild-type IL-17F., Conclusion: The current findings indicate that the IL-17F H161R variant influences the risk of asthma and is a natural IL-17F antagonist, suggesting a potential role for IL-17F in the etiology of asthma.

Oxidized DNA base lesions, such as thymine glycol (Tg) and 8-hydroxyguanine, are often toxic and mutagenic and have been implicated in carcinogenesis. To clarify whether NEIL1 protein, which exhibits excision repair activity towards such base lesions, is involved in gastric carcinogenesis, we examined 71 primary gastric cancers from Japanese patients and four gastric cancer cell lines for mutations and genetic polymorphisms of the NEIL1 gene. We also examined 20 blood samples from Chinese patients for NEIL1 genetic polymorphisms. Three mutations (c.82&#95;84delGAG:p.Glu28del, c.936G > A and c.1000A > G:p.Arg334Gly) and two genetic polymorphisms were identified. When the excision repair activity towards double-stranded oligonucleotide containing a Tg:A base pair was compared among six types of recombinant NEIL1 proteins, p.Glu28del-type NEIL1, found in a primary case, was found to exhibit an extremely low activity level. Moreover, c.936G > A, located in the last nucleotide of exon 10 and detected in the KATO-III cell line, was shown to be associated with a splicing abnormality using an in vivo splicing assay. An immunofluorescence analysis showed that the wild-type NEIL1 protein, but not the truncated protein encoded by the abnormal transcript arising from the c.936G > A mutation, was localized in the nucleus, suggesting that the truncated protein is unlikely to be capable of repairing nuclear DNA. An expression analysis revealed that NEIL1 mRNA expression was reduced in six of 13 (46%) primary gastric cancer specimens that were examined. These results suggest that low NEIL1 activities arising from mutations and reduced expression may be involved in the pathogenesis in a subset of gastric cancers.

L-Lysine dehydrogenase, which catalyzes the oxidative deamination of L-lysine in the presence of NAD, was found in the thermophilic bacterium Geobacillus stearothermophilus UTB 1103 and then purified about 3,040-fold from a crude extract of the organism by using four successive column chromatography steps. This is the first report showing the presence of a thermophilic NAD-dependent lysine dehydrogenase. The product of the enzyme catalytic activity was determined to be Delta1-piperideine-6-carboxylate, indicating that the enzyme is L-lysine 6-dehydrogenase (LysDH) (EC 1.4.1.18). The molecular mass of the purified protein was about 260 kDa, and the molecule was determined to be a homohexamer with subunit molecular mass of about 43 kDa. The optimum pH and temperature for the catalytic activity of the enzyme were about 10.1 and 70 degrees C, respectively. No activity was lost at temperatures up to 65 degrees C in the presence of 5 mM L-lysine. The enzyme was relatively selective for L-lysine as the electron donor, and either NAD or NADP could serve as the electron acceptor (NADP exhibited about 22% of the activity of NAD). The Km values for L-lysine, NAD, and NADP at 50 degrees C and pH 10.0 were 0.73, 0.088, and 0.48 mM, respectively. When the gene encoding this LysDH was cloned and overexpressed in Escherichia coli, a crude extract of the recombinant cells had about 800-fold-higher enzyme activity than the extract of G. stearothermophilus. The nucleotide sequence of the LysDH gene encoded a peptide containing 385 amino acids with a calculated molecular mass of 42,239 Da.

The most common form of hypersensitivity pneumonitis in Japan is summer-type hypersensitivity pneumonitis (SHP), which is caused by the inhalation of Trichosporon asahii or Trichosporon mucoides. To seek protein antigens relevant to the immunopathogenesis of SHP, we constructed a cDNA expression library of T. asahii, a major causative yeast species of SHP. Using the immunoscreening method, we identified and cloned a novel gene encoding a 19-kD protein, named TA-19, which proved to be specifically recognized in the bronchoalveolar lavage (BAL) fluids and sera of patients with SHP. IgG, IgA, and IgM antibodies to the recombinant TA-19 protein were significantly elevated in the sera as well as in the BAL fluids from SHP patients compared with those from non-SHP groups. This protein also induced SHP-specific proliferation of the mononuclear cells from both the peripheral blood and BAL. These results reveal that TA-19 derived from T. asahii may play a relevant role in specific cellular and humoral immune responses in patients with SHP.

Twenty-four genetic polymorphisms in the CYP2D6 gene were analysed in liver DNA samples of 39 Japanese and 44 Caucasians and compared with CYP2D6 protein levels and bufuralol 1'- and 6-hydroxylation activities in liver microsomes of these human samples. We detected 13 types of CYP2D6 genetic polymorphisms and classified these into 20 genotypes; nine types were found in Japanese and 14 types in Caucasian samples. CYP2D6*10B, but not CYP2D6*10A, was the most frequent (34.6%) in Japanese. In Caucasians, several CYP2D6 polymorphisms including CYP2D6*4, *4D, *4E, *4L, *3, *9, *5 and *2E (frequencies of 6.8, 3.4, 4.5, 9.1, 1.1, 2.3, 2.3 and 4.5%, respectively) were detected. A Caucasian having CYP2D6*3/*5 had a protein with slower gel mobility (immunoblotting with anti-CYP2D6 antibody) and very low activity for bufuralol 1'-hydroxylation. Five Caucasian samples (CYP2D6*4/*4, *4/*4L, or *4D/*4L) had no measurable CYP2D6 protein and very low bufuralol 1'-hydroxylation activities. Seven Japanese subjects with CYP2D6*10B/*10B had CYP2D6 protein at levels of approximately 20% of those present in humans with CYP2D6*1 and *2 and catalysed bufuralol 1'-hydroxylation at low rates. Kinetic analysis of bufuralol 1'- and 6-hydroxylation indicates that (i) the Km values for 1'-hydroxylation were lower in individuals with CYP2D6*1/*1, *1/*2, *1/*2X2, and *2/*2 than those with CYP2D6*4/*4, *4/*4L, *4D/*4L, or *10B/*10B and Vmax values tended to be higher in the former groups (*1, *2), and (ii) individuals with heterozygous CYP2D6*1/*4D, *1/*4L, and *1/*5 had relatively high Vmax/Km ratios, whereas individuals with heterozygous CYP2D6*1/*9, *2/4D, *2/*5, *2/*10B, *2E/*4E, *3/*5, *4L/*9, and *10B/*39 had lower Vmax/Km ratios for bufuralol 1'-hydroxylation. Quinidine inhibited bufuralol 1'-hydroxylation in liver microsomes, particularly at low substrate concentrations, in individuals with CYP2D6*1/*1, and 1/1*2, but not those with CYP2D6*4/*4 and very slightly in individuals with CYP2D6*10B/*10B. The latter two groups were found to be more sensitive to alpha-naphthoflavone than the former groups, indicative of the contribution of CYP1A2. These results support the view that CYP2D6*3, *4, *4D, and *4L are major genotypes producing poor metabolizer phenotypes in CYP2D6 in Caucasians, whereas CYP2D6*10B is a major factor in decreased CYP2D6 protein expression and catalytic activities in Japanese.

Chiba virus (CV), a Norwalk-like virus (NLV), was first identified as a cause of oyster-associated outbreak of gastroenteritis that occurred in Chiba prefecture, Japan, in 1987. An enzyme-linked immunosorbent assay (ELISA), based on hyperimmune antisera to recombinant baculovirus-expressed capsid proteins of CV (rCV), was developed to detect CV antigen in stools. No cross-reactions were observed with other enteric viruses including enteroviruses, rotaviruses, astroviruses, or enteric adenoviruses. The ELISA was used to screen 101 stools collected from 16 oyster-associated outbreaks of acute gastroenteritis. Twelve stools (11.9%) from seven outbreaks were positive for CV antigen. Ten rCV ELISA-positive strains were confirmed by RT-PCR and nucleotide sequencing. ELISA-positive strains showed 96-100% nucleotide sequence identity to each other, though they were obtained nine years apart. Phylogenetic analysis demonstrated that all ten strains clustered with the prototype CV in genogroup I viruses. We concluded that the antigen ELISA described in this study is highly type-specific, and that this method should be useful for epidemiological surveys of Chiba virus infections., (Copyright 2000 Wiley-Liss, Inc.)

Factor VII is a vitamin K-dependent zymogen that plays a key role in the initiation of the extrinsic pathway. A severe factor VII deficiency was identified in a 45-year old male whose plasma factor VII antigen was less than 60 ng/ml and expressed 5.2% of normal factor VII activity. DNA sequence analysis of the patient's factor VII gene showed a thymidine to guanine transversion at nucleotide 10968 in exon VIII that results in a novel amino acid substitution of His348 to Gln. The patient was homozygous for this mutation, whereas some of his family members were heterozygous. Both wild type and mutant factor VII were transiently expressed in COS-1 cells. The level of secreted mutant factor VII antigen was only 11.0% of the level of wild type factor VII. In CHO cells stably transfected with the mutant factor VII, only 37.3% of the total labeled FVII was secreted into the conditioned media and the remainder was retained inside the cells. These data suggest this mutation leads to factor VII deficiency due to the impaired secretion of the molecule.

The neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a food-borne botulism-related strain, Okra. The specific toxicity of 111/NT was found to be about 10 times lower than that of Okra/NT. The monoclonal antibodies recognizing the light chain cross-reacted with both neurotoxins, whereas most of the antibodies recognizing the carboxyl-terminal half of the heavy chain of Okra/NT did not react to 111/NT. Binding experiments with rat brain synaptosomes revealed that 125I-labeled 111/NT bound to a single binding site with a dissociation constant (Kd) of 2.5 nM; the value was rather lower than that (0.42 nM) of 125I-Okra/NT for the high-affinity binding site. In the lipid vesicles reconstituted with ganglioside GT1b, 125I-Okra/NT interacted with the amino-terminal domain of synaptotagmin 1 (Stg1N) or synaptotagmin 2 (Stg2N), fused with the maltose-binding protein, in the same manner as the respective full-length synaptotagmins, and the Kd values accorded with those of the low- and high-affinity binding sites in synaptosomes. However, 125I-111/NT only exhibited a low capacity for binding to the lipid vesicles containing Stg2N, but not Stg1N, in the presence of ganglioside GT1b. Moreover, synaptobrevin-2, an intracellular target protein, was digested to the same extent by the light chains of both neurotoxins in a concentration-dependent manner. These findings indicate that the 111/NT molecule possesses the receptor-recognition site structurally different from Okra/NT, probably causing a decreased specific toxicity.

Uridine monophosphate (UMP) synthase is a bifunctional enzyme catalyzing the last two steps of de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase (OPRT) and orotidine-5'-monophosphate decarboxylase (ODC). Loss of either enzymatic activity results in hereditary orotic aciduria, a rare autosomal recessive disorder characterized by retarded growth, anemia, and excessive urinary excretion of orotic acid. We have isolated the UMP synthase chromosomal gene from a lambdaEMBL-3 human genomic library and report a single-copy gene spanning approximately 15 kb. The UMP synthase genomic structure encodes six exons ranging in size from 115 bp to 672 bp, and all splicing junctions adhere to the canonical GT/AG rule. Cognate promoter elements implicated in glucocorticoid- and cAMP-mediated regulation as well as in liver-, myeloid-, and lymphocyte-specific expression are located within the 5' flanking sequence. Molecular investigation of UMP synthase deficiency in a Japanese orotic aciduria patient revealed mutations R96G (A-to-G transition; nt 286) and G429R (G-to-C transversion; nt 1285) in one allele and V109G (T-to-G transversion; nt 326) in the other allele. Expression of human UMP synthase cDNAs containing these mutations in pyrimidine auxotrophic Escherichia coli and in recombinant baculovirus-infected Sf21 cells demonstrates impaired activity presumably associated with the urinary orotic acid substrate accumulations observed in vivo. We further establish the identity of two polymorphisms, G213A (v = .26) and 440Gpoly (v = .27) located in exons 3 and 6, respectively, which did not significantly compromise either OPRT or ODC function.